Delivering nucleic acids into animal and plant cells has long been an important object of molecular biology research and development. Recent developments in the areas of gene therapy, antisense therapy and RNA interference (RNAi) therapy have created a need to develop more efficient means for introducing nucleic acids into cells.
A diverse array of plasmids and other nucleic acid “vectors” have been developed for delivering large polynucleotide molecules into cells. Typically these vectors incorporate large DNA molecules comprising intact genes for the purpose of transforming target cells to express a gene of scientific or therapeutic interest.
The process by which exogenous nucleic acids are delivered artificially into cells is generally referred to as transfection. Cells can be transfected to uptake a functional nucleic acid from an exogenous source using a variety of techniques and materials. The most commonly used transfection methods are calcium phosphate transfection, and electroporation. A variety of other methods for transducing cells to deliver exogenous DNA or RNA molecules have been developed, including viral-mediated transduction, cationic lipid or liposomal delivery, and numerous methods that target mechanical or biochemical membrane disruption/penetration (e.g., using detergents, microinjection, or particle guns).
RNA interference is a process of sequence-specific post transcriptional gene silencing in cells initiated by a double-stranded (ds) polynucleotide, usually a dsRNA, that is homologous in sequence to a portion of a targeted messenger RNA (mRNA). Introduction of a suitable dsRNA into cells leads to destruction of endogenous, cognate mRNAs (i.e., mRNAs that share substantial sequence identity with the introduced dsRNA). The dsRNA molecules are cleaved by an RNase III family nuclease called dicer into short-interfering RNAs (siRNAs), which are 19-23 nucleotides (nt) in length. The siRNAs are then incorporated into a multicomponent nuclease complex known as the RNA-induced silencing complex or “RISC”. The RISC identifies mRNA substrates through their homology to the siRNA, and effectuates silencing of gene expression by binding to and destroying the targeted mRNA.
RNA interference is emerging a promising technology for modifying expression of specific genes in plant and animal cells, and is therefore expected to provide useful tools to treat a wide range of diseases and disorders amenable to treatment by modification of endogenous gene expression.
There remains a long-standing need in the art for better tools and methods to deliver siRNAs and other small inhibitory nucleic acids (siNAs) into cells, particularly in view of the fact that existing techniques for delivering nucleic acids to cells are limited by poor efficiency and/or high toxicity of the delivery reagents. Related needs exist for improved methods and formulations to deliver siNAs in an effective amount, in an active and enduring state, and using non-toxic delivery vehicles, to selected cells, tissues, or compartments to mediate regulation of gene expression in a manner that will alter a phenotype or disease state of the targeted cells.